Figure 7.
Misfolded Wsc1p is not recognized by the major ER chaperone BiP/Kar2p. (A and B) Kar2p was immunoprecipitated from wild-type cells expressing Wsc1-L63R, Wsc1-L63RLuminal, or CPY* under native conditions. Protein complexes were resolved by SDS-PAGE and analyzed on immunoblots. Blots were probed using anti-HA antibody for substrates (top), stripped, and reprobed with anti-Kar2p antiserum as a control (middle). One percent of each lysate was loaded and analyzed in parallel to measure the relative amounts of input substrate used for each experiment (bottom). (C and D) Lysates from wild-type cells expressing the indicated substrates were subjected to native immunoprecipitation with anti-HA resin and Western blot analysis. Coimmunoprecipitated Kar2p was detected by anti-Kar2p immunoblots (top). The same blots were reprobed with anti-HA antibody as a control (middle). 1% of the lysates was analyzed in parallel and probed with anti-Kar2p antibody as an input control (bottom).